One of the most striking features of the lens, and one considered essential to optical clarity, is the exceptional degree of structural differentiation in fiber cells, and the precision and regularity of their tissue level organization. In the last funding period we established that the lens-specific Beaded Filament (BF), a derivative of the Intermediate Filament (IF) family, is required not to create these phenotypic features, but to maintain them with age. In the absence of the BF these unique structural feature are lost as the fiber cell and lens ages, result in loss of lens clarity. This application seeks to expand our knowledge of BF/IF structure, and to expand our focus from the filament, to the linker proteins and transmembane proteins that integrate the filament into the biology of the lens cell. We will look at Beaded Filaments and to a lesser extent Intermediate FiIaments. Toward these goals we propose to 1. Decipher the structure of the Beaded Filament and Intermediate Filament using a unique combination of Microscopic and Biochemical approaches, including Cryo Electron Microscopy and Site Directed Spin Labeling and Electron Paramagnet Resonance. 2. Identify linker proteins that couple the BF and IF to the fiber cell plasma membrane using Co- Immunoprecipitation, Yeast Two Hybrid Assays, Pull Down assays, ELISA Assays, immunocytchemical localization, and direct binding of recombinant linkers to BFs/IFs assembled in vitro from recombinant filament proteins. We will test biological function of linkers using CreLox conditional ablation of linkers 3. Identify transmembrane proteins, and membrane domains to which these filaments and linkers bind, and the membrane domains they form. We will use electron microscopy, co- immunoprecipitation assays, and Yeast Two Hybrid assays. PUBLIC HEALTH RELEVANCE We have identified two proteins that form a cytoskeletal support structure in cells of the ocular lens. Using genetic engineering approaches we have established that these proteins provide an "anti-aging" effect: They help cells resist the loss of structure that would otherwise occur with aging. In the absence of these proteins the lens undergoes a loss of organization that leads to a loss of optical clarity that gets progressively worse with age. [unreadable] [unreadable] [unreadable]